Worm Breeder's Gazette 13(2): 20 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Aiming to ultimately understand the network of gene expression in development of the worm, we are trying to construct an expression map of the 100Mb genome through identifying and characterizing all of its cDNA species, whose number is estimated to be around 15,000.
cDNA were made from mRNA of a mixed-stage population of him-8 strain and were size-fractionated before library construction. This time, a set of some 4,400 cDNA clones was selected from a library of >2kb cDNA by picking up cDNA clones (using lambdaZAPII as vector) at random and then removing very abundant cDNA clones (some 20 species which occupied ca. 20% of the library). cDNA inserts of individual clones were PCR-amplified using vector primers and then subjected to the analyses of (1) tag-sequencing, (2) mapping onto the genome, and (3) surveying expression pattern.
(1) Tag sequencing: Single reads have been made from both 5'-ends (using vector primers) and 3'-ends (using anchored oligo-dT primers in order to minimize the effect of long poly-A stretch) on ABI sequencers. Thus far, we have finished the tag-sequencing for almost all the clones. Out of them, 3,089 clones gave clean 3'-tag sequences, which were used to classify the clones into 1,478 species (808 standing-alone clones and 670 groups of 2-23 clones, see Fig. 1) by comparing the tags each other. This search has also detected pairs of clones which looked to be generated by alternative splicing. Database search showed that the set of 1,478 species hit 20% (36 genes) of C.elegans gene entries in GenBank and 23% of the clones gave significant similarities with genes of other organisms. At least 49 cDNA hit the 2Mb genomic region which has been sequenced by the genome sequencing consortium. Analysis of the 5'-tags are in progress.
(2) Mapping onto the genome: In order to make a lot of YAC polytene filters, we introduced a small press machine for dentists, which allowed us to easily print the YAC grid onto slave filters from a master; at least 300 replicas can be made from a single master. Although some clones could not be mapped using the Poly-2 filter, recently a supplementary filter (Suppoly) was made available (Thanks, Alan), with which most of the previously unmapped clones were mapped properly. Using these, about 700 cDNA species have been mapped onto the genome; central regions of chromosomes were much denser for cDNA than other regions.
(3) Surveying expression pattern: Since in situ hybridization analysis has confirmed the validity of the membrane strips on which aliquots of the 3'-end region of all cDNA species amplified from single embryos at various developmental stages have been dot-blotted (see Tabara & Kohara, in this issue), we are using the membrane strips for the surveying. Thus far, 126 cDNA species mapped on the chromosome 3 have been analyzed; briefly, 52 cDNA showed ubiquitous expression throughout embryogenesis, 19 looked maternal since they gave signals only on the dot of the Ohr embryo, 14 cDNA looked zygotic since they gave signals on the dots from later stage embryos but not from the Ohr embryo, the remainder gave no signals at all probably due to expression after embryogenesis or very low level of expression.
Part of these data are already in ACEDB version 2, and we are trying to send the remaining data to it as soon as possible. We are preparing cDNA filters (see below) and, of course, any individual clones are available.
As a next step, we have picked up altogether 30,000 cDNA clones at random from 3 different cDNA libraries; the same library of >2kb cDNA, a library of unfractionated cDNA, and an embryonic cDNA library. We are now making membrane blot of high-density grids of the clones, which will be used for probing with the already characterized cDNA to make a subset of the clones for unidentified cDNA species. We are going to continue the above analyses on the subset.